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*
Department of Nephrology and Medical Intensive Care,
Universitätsklinikum
Charité, Campus Virchow-Klinikum, Medizinische
Fakultät der
Humboldt-Universität zu Berlin,
Germany
Department of Pathophysiology, University Medical School, Poznan,
Poland
Fresenius Medical Care, Bad Homburg, Germany.
Correspondence to Dr. Achim Jörres, Department of Nephrology and Medical Intensive Care, Universitätsklinikum Charité, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany. Phone: +49 30 450 59318; Fax: +49 30 450 59918; E-mail: achim.joerres{at}charite.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In 1986, Henderson et al. suggested that abdominal pain experienced by patients receiving CAPD might be caused by irritant products of glucose degradation (13). The report has prompted research on the stability of glucose-based PDF and the potential toxicity of glucose derivatives. It has been demonstrated that breakdown of glucose contained in PDF gives rise to a number of products, several of which have been identified as low molecular weight aldehydes, including 5-hydroxymethyl-furfural, furaldehyde, acetaldehyde, formaldehyde, glyoxal, and methylglyoxal (14,15,16). The process occurs primarily during heat sterilization of PDF and to a lesser extent during their prolonged storage (17,18,19). It has been well documented that concentrations of GDP in heat-sterilized solutions are much higher than those detected in filter-sterilized fluids (20,21,22). The difference in biologic effects between heat- and filter-sterilized PDF has therefore been related—indirectly—to the presence of GDP. Indeed, in various experimental in vitro systems, heat-sterilized solutions have been shown to impair cell function to a greater extent than fluids sterilized by filtration (16, 20,21,22,23,24,25). The parameters that have been investigated included cell proliferation rate (15, 16, 20, 24, 25), cytokine synthesis (21, 22, 24, 26), phagocytosis (21), and superoxide generation (16, 21, 24). Using a different approach, Wieslander et al. have also demonstrated that several glucose-derived aldehydes were capable of directly inhibiting cell growth in culture (16). Most GDP studies to date have been performed with animal and/or transformed cells including mouse L929 fibroblasts (16, 20, 23, 25), RAW 264.7 macrophages (24), and human neuroblastoma SH-SY5Y cell line (24). Several studies have also used peripheral leukocyte subpopulations (21, 22, 24). However, very little is known about how exposure to GDP affects the function of peritoneal cells. The mesothelium forms the largest resident cell population in the peritoneal cavity. It is now well recognized that mesothelial cells not only form a barrier for the transport of solutes during peritoneal dialysis, but also play a crucial role in controlling the intraperitoneal inflammatory responses (27, 28). Thus, the potential detrimental effects of GDP toward mesothelial cells may impair intraperitoneal homeostasis in patients undergoing CAPD. In the present study, we have sought to examine whether the short-term exposure to GDP affects mesothelial cell functions, assessed as viability, proliferative capacity, and cytokine release.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Cultures
Human peritoneal mesothelial cells (HPMC) were isolated from the specimens
of omentum obtained from consenting nonuremic patients undergoing elective
abdominal surgery. Cells were isolated and characterized as described in
detail elsewhere (29). HPMC
were propagated in the M199 culture medium supplemented with L-glutamine (2
mM), penicillin (100 U/ml), streptomycin (100 µg/ml), hydrocortisone (0.4
µg/ml), and 10% vol/vol fetal calf serum (FCS) (Life Technologies,
Eggenstein, Germany). Cell cultures were maintained at 37°C in a
humidified atmosphere of 95% air and 5% CO2. All experiments were
performed using cells derived from at least five separate donors, and between
the first and third passage.
Mouse L929 fibroblast cell line was kindly provided by Dr P. Scholz (Schering, Berlin, Germany). Cells were maintained and propagated in the same medium as used for HPMC cultures.
Proliferation Studies
Cell proliferation was measured by [3H]-thymidine incorporation
assay. Cells were plated onto 48-well clusters at a density of 2.5 x
104/cm2 for HPMC or 1.0 x
104/cm2 for L929 fibroblasts, and cultured for 24 h.
Preliminary experiments had determined the optimal seeding density for each
cell type, which ensured that both populations were studied during the
exponential phase of growth. The subconfluent cell cultures were then exposed
to GDP in 10% FCS-containing medium and pulsed with [3H]-thymidine
(as methyl-[3H]-thymidine; 1 µCi/ml; Institute of Radioisotopes,
Prague, Czech Republic) for 24 h at 37°C. At the end of the exposure, the
labeling fluids were removed and the cells were detached with
trypsin:ethylenediaminetetra-acetic acid (0.05% wt/vol:0.02% wt/vol) solution
and precipitated with 10% (wt/vol) TCA. The precipitate was washed again with
10% TCA and finally dissolved in 0.1N NaOH. The released radioactivity was
measured in a beta liquid scintillation counter (LKB Wallac, Turku,
Finland).
HPMC Interleukin-6 Release
Induction of Interleukin-6 Production. HPMC were grown to confluence
in 24-well plates and rendered quiescent by serum deprivation. Briefly, HPMC
monolayers were transferred to the culture medium containing 0.1% FCS (rest
medium) for 48 h before stimulation. Previous experiments had demonstrated
that these conditions did not impair HPMC viability (as assessed by lactate
dehydrogenase [LDH] release and intracellular ATP levels)
(30). HPMC in the rest medium
were then exposed to GDP in the presence of recombinant human
interleukin-1ß (rhIL-1ß) (100 pg/ml; R&D Systems, Wiesbaden,
Germany) for 24 h at 37°C. After the incubation, the supernates were
removed, centrifuged at 12,000 x g to remove any cellular
debris, and stored at -70°C until assayed. Cell monolayers were washed
with Hanks' balanced salt solution (HBSS) and solubilized with 0.1N NaOH.
Total cellular protein was then analyzed with BCA protein assay (Pierce,
Rockford, IL), using bovine serum albumin as the standard. Repeated cell
counts revealed that 1 µg of HPMC protein corresponded to (mean ±
SD) 2.1 ± 1.0 x 103 cells (n = 16). All data
for interleukin-6 (IL-6) production were expressed as pg/µg cellular
protein.
IL-6 Measurements. IL-6 concentrations in HPMC supernates were measured with a specific "sandwich-type" immunoassay using an enzyme-linked immunosorbent assay-matched antibody pair (R&D Systems). The assay was designed and performed according to the manufacturer's instructions. Sensitivity of the system, determined by adding 2 SD to the mean optical density of the zero standard (n = 16), was 2 pg/ml.
Viability Studies
Release of LDH. After incubation under various experimental
conditions, the cell culture supernates were collected and assayed immediately
for LDH using a commercially available LDH kit (Analco-GBG, Warsaw, Poland).
Total intracellular LDH content was determined following the lysis of
representative cell monolayers with 0.1% (vol/vol) Triton X-100 in HBSS.
MTT Assay. The MTT assay is conventionally used for measuring cell proliferation. However, because the metabolic conversion of the MTT salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolinum bromide) is mediated by active mitochondrial dehydrogenases of living cells, the test can also be used for assessing cell viability (31). Briefly, after exposure to GDP, the cells were treated with MTT (1.25 mg/ml in culture medium) for 4 h at 37°C. The formazan product generated was solubilized by the addition of acidic solution of 20% (wt/vol) sodium dodecyl sulfate and 50% (vol/vol) N,N-dimethylformamide. Absorbance of the converted dye was recorded at 595 nm with a reference wavelength of 690 nm.
Effect of PDF
Test Solutions. To characterize the effect of GDP in the milieu of
PDF, the cells were exposed either to heat-sterilized PDF (H-PDF),
filter-sterilized PDF (F-PDF), or filter-sterilized PDF supplemented with a
known concentration of GDP (F-PDF + GDP). The fluids were prepared in the
laboratory according to the following formula (g/L): NaCl - 5.786,
CaCl2 x 2H2O - 0.257, MgCl2 x
6H2O - 0.102, sodium DL-lactate - 3.925, and anhydrous D-glucose -
15.0 or 42.5. The solutions from the same stock were then sterilized either by
heat (121°C, 0.2 MPa, 20 min) or filtration through 0.2-µm pore size
filter (Nalgene®, Nalge Nunc International, Rochester, NY). The extent of
glucose degradation was estimated by measuring the absorbance at 284 nm, which
is thought to reflect primarily the level of 5-HMF
(Figure 1) (22). The pH of all fluids
tested was adjusted to 7.3 with 0.1 M Na2CO3. The level
of endotoxin in all PDF preparations was <0.01 IU/ml as determined by
Limulus amoebocyte lysate assay using QCL-1000® Test kit
(BioWhittaker, Walkersville, MD). The doses of GDP applied corresponded to the
highest concentrations detected in PDF
(14): AcA 420 µM, FoA 15
µM, FurA 2 µM, Glx 14 µM, M-Glx 23 µM, and 5-HMF 30 µM.
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Exposure to PDF. Confluent cell monolayers of HPMC and L929 fibroblasts were pretreated with different types of PDF for specified times up to 3 h. Afterward, the test solutions were removed and replaced by fresh culture medium (with 0.1% FCS) in which cells were incubated for the next 18 h. After this recovery phase, the viability of cells was determined by LDH release and MTT test as described above. In parallel experiments, HPMC were incubated in the presence of IL-1ß (100 pg/ml) during recovery phase; supernates from these cultures were assayed for IL-6 while cell monolayers were solubilized and measured for protein contents.
Growth Inhibition Studies. In these experiments, the test solutions were prepared by mixing equal volumes of PDF and FCS-containing M199 medium labeled with [3H]-thymidine (final FCS concentration was 10%, and final [3H]-thymidine activity was 1 µCi/ml). In controls, the medium was mixed with HBSS to normalize for the dilution of culture medium components. HPMC or L929 fibroblasts in the exponential phase of growth were incubated in the presence of the PDF mixtures tested for 24 h, and the incorporated radioactivity was released as described above.
Statistical Analyses
All statistical analyses were performed using GraphPad PrismTM 2.00
software (GraphPad Software, San Diego, CA). Multiple comparisons of paired
data were made with nonparametric repeated-measures ANOVA with Friedman
modification. Data derived from mesothelial cells and L929 fibroblasts were
analyzed using Mann-Whitney U test for unpaired data and two-way
ANOVA. A P value <0.05 was considered significant. All data are
presented as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Viability
In HPMC treated with either FurA or 5-HMF, the release of LDH did not
differ from that in control cells. After exposure to all other GDP, the
release of LDH was augmented, but only when GDP were applied at the highest
concentrations tested (Figure
4A). FoA and M-Glx appeared to be most toxic, inducing a 5.7- and
2.6-fold rise in LDH release, respectively (n = 10, P <
0.001 and P < 0.01). This effect was accompanied by a simultaneous
decrease in the MTT conversion (Figure
4B). Although combinations of GDP did not alter the magnitude of
LDH release from HPMC (Figure
3C), both low and subtoxic doses of GDP reduced the conversion of
MTT by 15.3 ± 5.4 and 18.1 ± 4.5%, respectively (n = 9,
P < 0.05 for both) (Figure
3D). In contrast, neither individual nor combined GDP impaired the
viability of L929 fibroblasts as assessed by LDH release and MTT assay.
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HPMC IL-6 Synthesis
Incubation of HPMC in the presence of either AcA, FoA, Glx, or M-Glx
decreased IL-1ß-induced IL-6 production in a dose-dependent manner
(Figure 5). The greatest
inhibition was observed in cells treated with high concentrations of FoA and
M-Glx, in which the release of IL-6 was reduced by 85.1 ± 8.1 and 86.3
± 6.7%, respectively (n = 5, P < 0.05 for both).
Under the same conditions, FurA and 5-HMF did not affect IL-6 release by HPMC.
The levels of IL-6 recorded in HPMC cultures exposed to the mixtures of low
and subtoxic GDP doses were lower than those detected in the controls, and for
the subtoxic combination a reduction of 25.6 ± 18.0% reached
statistical significance (n = 7, P < 0.01)
(Figure 3B).
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Heat- versus Filter-Sterilized PDF
Effect of Short-Term Preexposure to PDF. Pretreatment of HPMC with
H-PDF at pH 7.3 and either at low (1.5%) or high (4.25%) glucose
concentrations resulted in a time-dependent reduction in IL-1ß-driven
IL-6 synthesis during the subsequent recovery period. This inhibition was
first evident in HPMC preexposed to 4.25%-H-PDF for 1 h, and after 2 h also in
cells pretreated with 1.5%-H-PDF. After a 3-h preexposure, the release of IL-6
was reduced from 17.9 ± 3.5 pg/µg cell protein in controls to 6.8
± 2.8 and 7.0 ± 2.8 pg/µg cell protein in HPMC incubated with
1.5%-H-PDF and 4.25%-H-PDF, respectively (n = 8, P < 0.05
and <0.001) (Figure 6). In
contrast, in HPMC treated with F-PDF, regardless of glucose concentration, the
secretion of IL-6 was not significantly diminished and remained above levels
detected in H-PDF-treated cells. Interestingly, the release of IL-6 from HPMC
preexposed to F-PDF supplemented with a defined mixture of GDP was not
different from that observed in cells treated with F-PDF alone.
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None of the PDF examined increased the release of LDH from HPMC during the period of direct exposure (data not shown). During the overnight recovery phase, however, the LDH release became elevated in all cultures treated with PDF containing 4.25%, but not 1.5%, glucose (Figure 7A). This increase was significantly above background levels after a preexposure period of 3 h, with no differences observed between various types of 4.25%-PDF tested. This pattern of impaired HPMC viability was also recorded in the MTT assay (Figure 7B). In cells pretreated with H-PDF, F-PDF, or F-PDF + GDP (all with 4.25% glucose), the conversion of MTT was reduced to 71.4 ± 13.1% (P < 0.001), 79.6 ± 13.3% (P < 0.05), and 78.2 ± 11.5% (P < 0.05) of the control level, respectively (n = 10). In contrast to HPMC, the viability of L929 fibroblasts—assessed both in the LDH test and in the MTT assay—was not affected by the preexposure to PDF of any type.
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Inhibition of Cell Growth in Response to PDF. The FCS-stimulated proliferation of HPMC was significantly reduced in the presence of autoclaved PDF, with solutions mixed with 4.25%-PDF being more inhibitory than those supplemented with 1.5%-PDF (Figure 8). The degree of inhibition exerted by these solutions was significantly greater than that of their filtered counterparts (66.6 ± 6.0% versus 31.9 ± 8.7% and 87.2 ± 3.2% versus 51.3 ± 7.8% of inhibition for 1.5%-and 4.25%-PDF, respectively; n = 10, P < 0.05 for both). However, the smaller inhibitory potential of F-PDF could be substantially augmented by the addition of a defined mixture of GDP (the differences between GDP-spiked F-PDF and F-PDF alone were not formally significant when compared by ANOVA, but the same test revealed no differences between F-PDF + GDP and H-PDF).
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The pattern of growth inhibition in L929 cells was very similar. Although the magnitude of inhibition in response to H-PDF did not differ from that observed in HPMC, the solutions sterilized by filtration (also with additional GDP) appeared to be less inhibitory in L929 fibroblasts than in HPMC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have found that none of the GDP studied impaired the viability and function of HPMC over a 24-h period if applied individually at low concentrations that approximated those seen in PDF. The addition of higher GDP doses, however, led to the significant suppression of HPMC functions with marked differences between various compounds examined. FoA, Glx, and M-Glx appeared to be most toxic, causing a profound dose-dependent reduction in both HPMC proliferation and IL-6 synthesis. These inhibitory effects could be partially attributed to direct HPMC injury because either FoA, Glx, or M-Glx at high doses caused a significant loss of HPMC viability as assessed by LDH release and MTT test. Adverse effects of AcA and FurA were detected only in [3H]-thymidine incorporation assay, while 5-HMF had no significant impact on any functional parameter studied. The level of 5-HMF in glucose-containing solutions is often regarded as a general indicator of glucose breakdown (14, 22, 44). The fact that 5-HMF does not seem to be acutely toxic to HPMC over a vast range of concentrations suggests that the assessment of PDF biocompatibility based solely on 5-HMF measurements may overlook the impact of other more toxic GDP. Another glucose derivative that is thought to better reflect the magnitude of glucose degradation is acetaldehyde (20, 21). In this respect, it was interesting to observe that AcA decreased HPMC proliferation already at a dose of 10 µg/ml, the level that could be reached in some H-PDF (16). The same dose, however, neither diminished IL-1ß-stimulated IL-6 production nor damaged cell membrane integrity as judged by LDH release. These observations indicate that in assessing GDP-associated toxicity, it is necessary to examine more than one parameter because cell functions may—contrary to previous suggestions (22)—display different sensitivity to GDP action.
Although individual GDP at low doses did not exert significant effects on HPMC, one cannot rule out that, when combined, GDP may act in a synergistic manner. Indeed, a combination of low GDP doses appeared to interfere with the function of HPMC mitochondria as demonstrated by a slightly reduced metabolic conversion of MTT. This synergistic effect occurred more clearly at higher levels of concentrations. Exposure of HPMC to GDP combined together at maximal doses, which proved noninhibitory when tested separately, produced a detectable decrease in cell proliferation and IL-1 ß-driven IL-6 synthesis. This subtoxic combination contained higher concentrations of Glx, 5-HMF, and FurA compared with the low-dose GDP mixture. Interestingly, we were unable to demonstrate any damage to cell membrane integrity under these conditions as judged by LDH release. In this respect, we have previously shown that short-term exposure of HPMC to dialysis solutions may impair the mitochondrial generation of ATP without increasing LDH release (30).
In general, our results are in agreement with those reported previously by Wieslander et al., who found that the same glucose-derived aldehydes at doses detected in PDF did not impair the proliferation of murine L929 fibroblasts (16). This lack of significant inhibition was evident even in cells exposed to GDP for as long as 72 h compared with 24 h in our system. To evaluate differences in HPMC and L929 fibroblast responses to GDP more precisely, we have analyzed their growth rates in the presence of GDP using the same type of culture medium, the same FCS batch, and identical exposure times. Preliminary experiments revealed that L929 fibroblasts could be easily maintained in the M199 HPMC-specific medium rather than in the MEM medium used in other studies (16, 20, 25). Growth responses of L929 cells recorded under these conditions confirmed the results of Wieslander's group in terms of either the character or magnitude of induced changes (16). However, as demonstrated by two-way ANOVA, AcA, Glx, and FurA suppressed the proliferation of L929 cells to a significantly smaller degree compared to HPMC. Lack of such differences in cells exposed to other aldehydes could be explained either by extreme toxicity occurring within a relatively narrow range of doses (FoA, M-Glx) or, conversely, by the absence of significant impact on cell proliferation (5-HMF). These results may suggest that HPMC are more susceptible to the insult from GDP. Indeed, we were unable to detect GDP-mediated injury to L929 cells, as measured by LDH release or MTT assay, even with the doses that were clearly cytotoxic for HPMC or any GDP combination.
It has been assumed that high GDP concentration is responsible for the disadvantageous biocompatibility characteristics of H-PDF. We have therefore compared the function of cells preexposed to differently sterilized PDF. We have found that the inhibition in IL-6 release from HPMC preexposed to H-PDF was greater than that caused by F-PDF. However, HPMC viability was reduced to a similar degree regardless of the mode of sterilization and the presence of additional GDP. Importantly, none of the PDF studied impaired the viability of L929 fibroblasts in this system. Because it could be argued that the exposure period of 3 h was not long enough for GDP to exert a more specific impact on HPMC, in the next series of experiments we have exposed cells for up to 24 h to PDF diluted in pH neutral, FCS-supplemented medium, as described originally by Wieslander et al. (25). In this setting, and in line with several previous reports (21, 22, 24, 25), we recorded a significantly greater degree of inhibition in response to H-PDF compared with F-PDF. We have even been able to demonstrate a significant difference between proliferation rates of HPMC treated with H- and F-PDF of 1.5% glucose concentration, while in a recent report it has been suggested that in low glucose-based PDF, the levels of GDP might be too small to produce a distinction in biologic responses and that any significant difference between H- and F-PDF could only be detected in solutions with high (3.86%) glucose concentration (20). Although the difference in HPMC reaction to heat- and filter-sterilized fluids is in agreement with observations in other cell types, the extent of this response is somewhat surprising considering the fact that GDP at corresponding doses added to culture media had a very limited impact on HPMC proliferation. One may hypothesize that other factors present in PDF may influence the action of GDP. In the present study, we chose to examine GDP at neutral pH to exclude well-known inhibitory effects associated with acidic pH. However, it has recently been suggested that in the in vivo situation the combination of GDP and low pH may in fact accentuate the effects of GDP as demonstrated by changes in small solute transport during peritoneal dialysis in rats (45). To confirm the impact of GDP in the milieu of PDF, we exposed HPMC to F-PDF supplemented with the highest GDP concentrations detected in autoclaved PDF. The combination of F-PDF + GDP appeared to be more inhibitory toward HPMC proliferative capacity than F-PDF alone. This observation indicates that the six aldehydes tested in the present study may contribute, at least in part, to detrimental effects produced by PDF in vitro. In contrast, Wieslander et al. failed to demonstrate that aldehyde-spiked F-PDF differed in ensuing biologic effects from PDF without additional GDP (16). It is possible that the overall effects attributed to GDP are also mediated by the relative presence of different, not-yet-defined or quantified compounds. For example, other glucose derivatives that may be detected in PDF include formic acid, valeraldehyde (14), and 3-deoxyglucosone (46), and their impact on HPMC function remains to be elucidated.
The response of L929 fibroblasts to H-PDF was very similar to that of HPMC. In view of earlier results, this observation was rather unexpected and might indicate that proliferating L929 fibroblasts are much more vulnerable to GDP (and GDP-containing PDF) than the confluent L929 monolayers. On the other hand, greater inhibition of HPMC proliferation after exposure to F-PDF suggests that HPMC are more sensitive to PDF components other than GDP. Because the L929 cell line provides a well-characterized and reproducible cell system that is commonly used for cytotoxicity testing, these considerations should be taken in account when examining effects of GDP and/or PDF. Our data indicate that under certain experimental conditions, the exclusive use of L929 cells may underestimate the full extent of GDP-associated toxicity, and for this purpose it may be more appropriate to use primary cell cultures of peritoneal origin.
Taken together, our results indicate that GDP possess a significant cytotoxic potential toward HPMC. During the clinical setting of CAPD, mesothelial cells are continually exposed to GDP for extended periods of time. It is therefore possible that GDP exert cumulative effects on long-term cell function rather than produce direct acute injury to HPMC. The potential of GDP to evoke chronic HPMC dysfunction is currently being investigated. Mechanisms by which GDP may affect long-term HPMC functions are poorly defined. In recent years, it has become increasingly evident that GDP (M-Glx and 3-deoxyglucosone in particular) are potent promoters of advanced glycosylation end product (AGE) formation (47,48,49). It has been suggested that a considerable capacity of glucose-based PDF for AGE formation is related to the presence of GDP rather than glucose per se (50, 51). In this respect, Nakayama et al. have clearly documented the accumulation of AGE in the peritoneum of CAPD patients (52). It is also possible that the biologic impact of GDP is influenced by the uremic environment. Increased levels of 3-deoxyglucosone and glyoxal- and methylglyoxal-mediated protein cross-links have been reported in sera of uremic patients (53,54,55). It has also been demonstrated that methylglyoxal-modified proteins undergo a receptor-mediated endocytosis and stimulate production of inflammatory cytokines in mononuclear cells (56,57,58,59). Whether these mechanisms are involved in long-term effects of GDP on HPMC remains to be determined.
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Acknowledgments |
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This work was supported by a grant from the Else Kröner-Fresenius Foundation (Bad Homburg, Germany).
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Footnotes |
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References |
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) and L929 cells (
) exposed to
increasing doses of GDP for 24 h. Data are expressed as a percentage of
[3H]-thymidine incorporation into control cells incubated in the
absence of GDP. Data represent the mean ± SEM of five experiments (with
HPMC isolated from different donors). *, statistically significant
difference compared to the respective control; #, difference in relative
growth inhibition between L929 cells and HPMC.
P <
0.05 compared to heat-sterilized PDF of respective glucose concentration;
#P < 0.05 L929 versus HPMC.